PROJECT SUMMARY/ABSTRACT Although Zika virus (ZIKV) was discovered almost 70 years ago in Zika forest in Uganda, it is only recently recognized as a global public threat. Historically, ZIKV infection has been associated with asymptomatic illnesses such as rash, arthralgia, and conjunctivitis. However, recently several studies have found that ZIKV infection causes severe fetal abnormalities that include microcephaly in infants and the neurological disorder GBS in adults. To date, there is no vaccine or antiviral therapy, and limited information is available for accurately predicting the outcome as well as the resulting complications due to ZIKV infection. There is a knowledge gap in understanding the basic pathogenesis of this virus, which is critical for developing strategies to combat this disease. The role of host factors especially that of host lipids in ZIKV pathogenesis is largely unknown. The profound success of flaviviruses such as ZIKV in causing disease depends on its ability to successfully utilize the host's cellular machineries including the lipid biosynthesis pathway to subvert the immune system. Our understanding of these processes especially that of the host lipid biology in the context of ZIKV is limited. The aim of the proposed work is to study the role of host's lipid biosynthesis in the entry, replication, assembly and release of ZIKV. For this purpose, individual knockout Huh7 and SH-SY5Y cell lines that lack key genes involved in the biosynthesis of lipids will be generated using CRISPR/Cas9-technology. To complement our genetic approach, small molecule inhibitors will be used to block these key pathways, and their impact in ZIKV infection will be investigated. In addition to plaque assay, automated high-content microscopy will be performed in ZIKV infected mutant and control cells to define the function of host lipids at different stages of ZIKV infection. One of the major cellular antimicrobial processes, autophagy, will be assessed in both knockout and control cells during ZIKV infection. Using lipid reporter tools we also plan to investigate the spatial and temporal (re-)distribution of lipids during ZIKV infection. Understanding these pathways or processes essential for the life cycle of ZIKV is crucial, as they represent potential targets for new therapeutics.